P. aeruginosa is one of the principal pathogens associated with Cystic fibrosis (CF) pulmonary infection and ranks 2nd among the most common pathogens isolated from chronic and burn wounds. Once established, P. aeruginosa biofilms are difficult to eradicate by antimicrobial treatment. While the clinical relevance of biofilms is well established, little is known about the mechanism of biofilm dispersion, the last step in biofilm development. We hypothesize that biofilm dispersion occurs via modulation of c-diGMP levels, protein-protein interactions, and protein phosphorylation. We also hypothesize that biofilm dispersion alters the virulence of P. aeruginosa in acute and chronic infections. Recent findings indicate that P. aeruginosa biofilm dispersion coincides with a switch in phenotype and a decrease in the intracellular signaling messenger cyclic-di-GMP (c- diGMP). Evidence from our lab also indicates that dispersion requires protein phosphorylation and synthesis. Furthermore, biofilm dispersion has been shown in our lab to be regulated by at least seven proteins including BdlA, 2 sensor proteins, potential receptors for nutrient-induced dispersion signals, proteins involved in reciprocally modulating c-diGMP levels, and a response regulator, that we believe form part of a novel pathway that is distinct from known c-diGMP-modulated pathways. Our data further indicate that dispersion-deficient P. aeruginosa mutants are less virulent. The goal of the proposed studies is to characterize the pathway involved in regulating biofilm dispersion, and to determine the role of dispersion in acute and chronic infections. We will use in Specific Aim 1 in vivo and in vitro assays to determine which of the proteins have c-diGMP modulating activity and whether their enzymatic activity is dependent on BdlA. In Specific aim 2, we will make use of V5- His-fusions and co-immunoprecipitation ("pull-down") assays in the presence of crosslinking agents to determine whether protein-protein interactions play a role in biofilm dispersion. Protein identifications will be used in combination with information on protein-protein interactions to determine direct, convergent or indirect pathways involved in dispersion. In specific Aim 3, we will determine the role of phosphorylation in dispersion and modulation of c-diGMP enzymatic activities using biochemical, genetic and proteomic approaches. In Specific Aim 4, we will characterize the role of BdlA and three other proteins in P. aeruginosa virulence and biofilm formation in vivo using chronic and acute lung infection models. The experiments are designed to determine whether P. aeruginosa mutants impaired in biofilm dispersion are less virulent in acute infections but more virulent in chronic infection. Assessments will be based on bacterial enumeration, lung histopathology, and biophotonic image scanning which allows for the visualization of bacterial dissemination and biofilm formation. Findings from this detailed investigation of the dispersion process are expected to lead to more effective treatment strategies based on inhibition or regulation of biofilm dispersion to treat and control biofilm infections. PUBLIC HEALTH RELEVANCE: Statement Pseudomonas aeruginosa is a common cause of hospital acquired infection and the leading cause of death in patients with cystic fibrosis. One of the hallmarks of P. aeruginosa is its high intrinsic resistance to antibiotics. New strategies are therefore needed to combat this bacterium. This proposal is aimed at understanding the role of the sensor protein BdlA in virulence properties of P. aeruginosa in chronic and acute infection models and in the pathway resulting in biofilm dispersion. Findings from this research are anticipated to result in innovative and effective treatment strategies based on inhibition or regulation of genes involved in biofilm dispersion to prevent systemic infections and/or to treat and control biofilm infections.